CRISPR 101 | Everything You Need to Know About CRISPR
A bacterial immune system became the world’s best genome engineering tool and it can treat HIV.
CRISPR seems like a technology out of a sci-fi movie.
Nevertheless, the bacterial immune system first observed 27 years ago allows us to manipulate the human genome in ways that even sci-fi movies had not predicted.
The discovery of CRISPR and its function was made in 1993–2005 by Francisco Mojica. The discovery of Cas9 and PAM was made in 2005 by Alexander Bolotin.
Twenty-seven years later and the Nobel Prize winners in Chemistry 2020 are Jennifer Doudna and Emmanuelle Charpentier’, the two scientists who pioneered the CRISPR system for genome engineering.
The genome engineering tool can treat genetic disorders and autoimmune disorders while creating change in agriculture. One example of such advancement is CRISPR’s use to develop stem cell therapeutics for the Human Immunodeficiency Virus (HIV).
Table of Contents:
CRISPR as an Immune System in Bacteria
Using CRISPR for Gene Knockout
Solving Real World Problems with CRISPR
CRISPR as an Immune System in Bacteria
The CRISPR system is an immune system in bacteria against viruses, specifically bacteriophages, used today by scientists for genome engineering. Bacteriophage is a virus that infects bacteria and inserts its DNA into the bacteria.
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeat. Scientists created this name when they observed that segments of bacterial DNA had repeated palindromic bases.
Palindromic means that a number or a word — such as civic or kayak — reads the same if you read it from the left or right. On a DNA sequence, a palindromic base sequence would look something like GTG or ACA. The palindromic sequences are called repeats.
However, scientists observed that the base sequences in between these palindromic repeats were all different. These in-between sequences were called spacers. Scientists concluded from this CRISPR array that the viral DNA is stored in between the repeats as spacers.
How did the spacers get inserted in the CRISPR array? The answer lies in the simple yet effective immune system of bacteria.
When the viral DNA from the phage attempts to invade the bacterial DNA, the bacteria cleave segments of approximately 20bp of the viral DNA using Cas endonucleases and integrates it into its genome. The DNA segment becomes a novel spacer incorporated into the CRISPR array.
If the phage tries to attack again, the bacteria have the viral DNA memorized in its genome. Cas proteins transcribe CRISPR RNAs (crRNA). The nitrogenous bases used for RNA are Adenine, Cytosine, Guanine, and Uracil.
The crRNA integrates with a protein called Cas9, an endonuclease that acts as a molecular scissor, creating a cascade complex. The Cas9 protein is one of the Cas proteins synthesized by the Cas genes.
Through sequence homology, the crRNA guides Cas9 to the exogenous genetic material — the viral DNA — to unwind the DNA and cleave it. A double-stranded break where both helices break and the phage is disabled.
The exogenous genetic material must have a protospacer adjacent motif (PAM) directly after the location where the crRNA will bind. PAM is a sequence of 2–6 base pairs located 3–4 nucleotides downstream of the target sequence — also called the protospacer.
Using CRISPR for Gene Knockout
The CRISPR-Cas9 system can knock out genes of ~20 bases in mammalian cells with the co-expression of a Cas endonuclease and a gRNA for the target gene.
However, we can only knock out a gene if its sequence is unique compared to the rest of the genome and adjacent to a PAM. It is vital to select a Cas protein from a species with your desired PAM sequence.
The system is composed of two parts:
1) gRNA
2) Cas endonuclease (Cas9 and Cpf1 are the most commonly used)
Firstly, gRNA is composed of crRNA (5' end) and tracrRNA (3' end) that have been linked together in the lab by a linker loop. This structure is called a Chimera. The crRNA is called a spacer, and the tracrRNA, together with the linker loop, is called a scaffold.
Secondly, the Cas endonuclease attaches to the gRNA through positively-charged grooves exposed on the Cas endonuclease surface. A ribonucleoprotein complex (also known as a cascade) is created.
The interaction between the gRNA and the Cas endonuclease shifts the Cas molecule from being inactive non-DNA binding to active DNA-binding, allowing the Cas molecule to cleave the DNA. When the Cas endonuclease binds to the DNA strand, the spacer region of gRNA remains free to interact with the target sequence.
If we assume that we are using Cas9 as an endonuclease, then the Cas9-grNA complex will bind with the DNA sequence with the most homology. The seed sequence, 8–10 bases on the 3' end of the gRNA, anneals to the target sequence. If the target sequence and gRNA match each other, the rest of the gRNA will anneal the DNA from the 3' end to the 5' end.
The opposite strands of the target sequence are cleaved by the nuclease domains RuvC and HNH of the Cas9 that has gone through a conformation. A double-stranded break gets created on the DNA.
The double stranded break repaired in two ways:
1) Non-homologous end joining (NHEJ)
2) Homology directed repair (HDR)
NHEJ is fast yet random. It creates small insertions and deletions in the target DNA that cause deletions, insertions and frameshifts in the amino acids. The open reading frame of the gene gets disrupted. Consequently, NHEJ is not a precise repair method, but it is the most common repair method when the Cas9 endonuclease has created a double-stranded break.
HDR allows precise insertion and deletion for nucleotides. A DNA repair template containing the desired DNA sequence is introduced to the cell by the Cas9-gRNA complex. Two more DNA repair templates are introduced on the right and left of the target sequence: these are called the right and left homology arms.
However, the efficiency of HDR is low. One area of research to fix this problem is by synchronizing the cells during the cell cycle.
Solving Real World Problems with CRISPR
CRISPR is one of the most programmable, specific and cheapest genome engineering tools available on the market as the newest disruptive technology in the field.
From being used in agriculture to increase plant yield, resistance to diseases and herbicides and creating germplasms with desirable traits (Zhu, Li, Gao, 2020) to using CRISPR for in vivo therapeutics to treat genetic disorders, the applications of CRISPR are endless.
CRISPR to Treat HIV-1
In October 2020, Jenna Kropp Schmidt, Nick Strelchenko, and Mi Ae Park released their research on “Genome editing of CCR5 by CRISPR-Cas9 in Mauritian cynomolgus macaque embryos”. This research demonstrates the use of CRISPR in stem cell based HIV therapeutics.
Resistance to the HIV-1 virus in humans is associated with homozygous 32 base pair deletion in the CCR5 gene. The deletion results in the cell losing the activation of the CCR5 co-receptor, allowing the viral fusion to the cell membrane, allowing viral content into the cell. A possible cure to HIV is using CCR5-mutant (CCR5-delta32) hematopoietic stem cells (HSCs).
Macaques infected with SIV are great animal models to study HIV-1 in humans because they have a high degree of MHC allele sharing. MCM embryos were grown in vitro, and the CCR5 deletion was administered to the embryo using a dual-guide gene targeting approach.
The results:
“Biallelic deletions in the CCR5 gene were introduced into ~ 23–37% of MCM embryos.”
“single blastomere PCR analysis revealed mosaicism in CCR5 editing within the same embryo”
“Despite relatively low embryonic development rates, successful CRISPR-Cas9 targeting of CCR5 was achieved in MCM embryos.”
The implications of this research:
CRISPR can delete genes associated with HIV in stem cells as a therapy for HIV patients.
Read the full research paper here.
citation: Schmidt, J.K., Strelchenko, N., Park, M.A. et al. Genome editing of CCR5 by CRISPR-Cas9 in Mauritian cynomolgus macaque embryos. Sci Rep 10, 18457 (2020).
Key Takeaways
- The Discovery of CRISPR was 27 years ago.
- CRISPR is a bacterial immune system involving a cascade composed of a crRNA and Cas endonuclease that cleaves viral DNA.
- Knocking out genes with CRISPR is possible by combining a gRNA and Cas endonuclease. Additionally, a specific PAM sequence must be determined.
- CRISPR is programmable, specific and cheap.
- Researchers are using CRISPR to delete the homozygous 32 base pair in the CCR5 gene to create Stem cell therapies for HIV.
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Additional Resources
Learn more about the history of CRISPR.
